CUT&RUN and HiChIP of CTCF and histone modifications in Wnt ON and Wnt OFF in HEK293T cells
CUT&RUN and HiChIP of CTCF and histone modifications in Wnt ON and Wnt OFF in HEK293T cells
RUW regions were amplified from genomic DNA of transfected populations via PCR with the high fidelity Q5 polymerase (New England Biolabs, Cat. # M0491S), with an annealing temperature of 62 degrees and performing 35 cycles.  HEK293T human embryonic kidney cells (parental) and Δβ-catenin cells (generated in PMID: 30425074) were cultured in a 37 °C incubator in 5% CO2 and 89% humidity. Culture medium used was high glucose Dulbecco's Modified Eagle Medium (Cat. #41965039, Gibco) supplemented with 10 % bovine calf serum (Cat. #1233C, Sigma-Aldrich) and 1X Penicillin-Streptomycin (Cat. #15140148, Gibco).   Cells were stimulated either with 10 µM CHIR99021 (Wnt ON) or 1 nM LGK (Wnt OFF) for 24 hr prior to harvest for assays.  Correct product size and a single product was confirmed with an agarose gel, then the DNA was purified with bead cleanup to remove primers and contaminants. Library preparation was performed according to PMID: 36355069 using the KAPA Hyperprep Kit (KAPA Biosystems, Cat. #KK8504) with the following modifications: for CTCF samples, 15 cycles of amplification were performed, while LoV-U samples received 13 cycles as previously described. For CTCF samples, size selection was performed post-amplification using 2% E-Gel Size Select II Gels (Thermo Fisher, Cat #G661012), retrieving fragments from 150 – 240 bp (representing 30 – 120 bp without the adapter sequences).